Inhibition of endosomal toll-like receptor activation

ABSTRACT

The present invention relates, in general, to pattern-recognition receptors (PRRs), including toll-like receptors (TLRs), and, in particular, to a method of inhibiting nucleic acid-induced activation of, for example, endosomal TLRs using an agent that binds to the nucleic acid (“nucleic acid binding agent”), preferably, in a manner that is independent of the nucleotide sequence, the chemistry (e.g., DNA or RNA, with or without base or sugar modifications) and/or the structure (e.g., double-stranded or single-stranded, complexed or uncomplexed with, for example protein) of the nucleic acid(s) responsible for inducing TLR activation. The invention also relates to methods of identifying nucleic acid binding agents suitable for use in such methods.

This application is a U.S. National Phase of International ApplicationNo. PCT/US2010/002516, filed Sep. 16, 2010, which designated the U.S.and claims priority from U.S. Provisional Application No. 61/243,090,filed Sep. 16, 2009, the entire contents of each of which areincorporated herein by reference.

This invention was made with government support under Grant No. HL65222awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to pattern-recognitionreceptors (PRRs), including toll-like receptors (TLRs), and, inparticular, to a method of inhibiting nucleic acid-induced activationof, for example, endosomal TLRs using an agent that binds to the nucleicacid (“nucleic acid binding agent”), preferably, in a manner that isindependent of the nucleotide sequence, the chemistry (e.g., DNA or RNA,with or without base or sugar modifications) and/or the structure (e.g.,double-stranded or single-stranded, complexed or uncomplexed with, forexample protein) of the nucleic acid(s) responsible for inducing TLRactivation. The invention also relates to methods of identifying nucleicacid binding agents suitable for use in such methods.

BACKGROUND

TLRs are type I transmembrane proteins composed of an extracellulardomain of leucine-rich repeats and an intracellular Toll/interleukin-1(IL-1) receptor (TIR) domain (Leulier and Lemaitre, Nat. Rev. Genet.9:165-178 (2008)). Ten human and twelve mouse TLRs have been identified.Each TLR is able to recognize a particular molecular pattern. Forinstance, TLR2, 4, 5, 6 and 11 bind to bacterial outer membranemolecules such as lipopolysaccharide (LPS), peptidoglycan and lipoteicacid while TLR3, TLR7, TLR8 and TLR9 recognize bacterial, viral or evenendogenous nucleic acids (Kawai and Akira, Semin. Immunol. 19:24-32(2007)). Moreover, TLRs can be classified based on their cellularlocalization: TLR1, 2, 4, 5 and 6 are expressed on the cell surfacewhile TLR3, 7, 8 and 9 are localized mostly, though not exclusively, inendosomal compartments (Kawai and Akira, Semin. Immunol. 19:24-32(2007)).

When pathogens invade a host, innate immune cells such as macrophages,neutrophils, natural killer cells and dendritic cells recognizepathogen-associated molecular patterns (PAMPs) and endogenousdamage-associated molecular patterns (DAMPs) through TLRs. TLRactivation initiates intracellular signaling events that result in theexpression of immune response genes including inflammatory and immunemodulatory cytokines, chemokines, immune stimulatory receptors, whichaugments killing of pathogens and initiates the process of developingacquired immunity (Takeda and Akira, Int. Immunol. 17:1-14 (2005), Akiraet al, Cell 124:783-801 (2006)). Inappropriate activation of somemembers of the TLR family, on the other hand, contribute to developmentof a variety of diseases including bacterial sepsis (TLR1, TLR2, TLR3,TLR4 and TLR9) (Wurfel et al, Am. J. Respir. Crit. Care Med. 178:710-720(2008), Knuefermann et al, Circulation 110:3693-3698 (2004), Cavassaniet al, J. Exp. Med. 205:2609-2621 (2008), Alves-Filho et al, Crit. CareMed. 34:461-470 (2006), Tsujimoto et al, J. Hepatol. 45:836-843 (2006)),non-infection systemic inflammatory response syndrome (TLR4) (Breslin etal, Shock 29:349-355 (2008)), multiple sclerosis (TLR3, TLR4 and TLR9)(Chen et al, Int. Immunopharmacol 7:1271-1285 (2007)), systemic lupuserythematosus (SLE) (TLR7 and TLR9) (Marshak-Rothstein and Rifkin, Annu.Rev. Immunol. 25:419-441 (2007)) and rheumatoid arthritis (TLR3, TLR4,TLR7, TLR8 and TLR9) (Choe et al, J. Exp. Med. 197:537-542 (2003),O'Neil, Nat. Clin. Pract. Rheumatol. 4:319-327 (2008)). Moreover,preclinical and clinical studies indicate that inhibition of TLRactivity has therapeutic benefits for treating certain diseases. Forexample, diverse LPS-neutralizing agents and TLR4 antagonists have beenevaluated to treat inflammatory diseases in animal and clinical studies(Leon et al, Pharm. Res. 25:1751-1761 (2008)). A TLR9 inhibitor,inhibitory CpG DNA (Plitas et al, J. Exp. Med. 205:1277-1283 (2008)),and an antagonistic anti-TLR3 antibody (Cavassani et al, J. Exp. Med.205:2609-2621 (2008)) enhanced survival of a mouse with polymicrobialsepsis. Oligonucleotide-based TLR7 and TLR9 inhibitors prevented IFNαproduction from human plasmacytoid dendritic cells stimulated with serumfrom SLE patients (Barrat et al, J. Exp. Med. 202:1131-1139 (2005)).Unfortunately, the redundancy of the TLR family may limit the utility ofinhibitors that target individual TLRs.

Upon stimulation, all TLRs recruit intracellular TIR-domain-containingadapters, such as TRIF and MyD88 (Kawai and Akira, Semin. Immunol.19:24-32 (2007)). These adapter molecules mediate a downstream cascadeof TLR-associated signaling. TRIF is recruited to TLR3 and TLR4, andappears to activate IRF3, MAPK, and NF-κB while MyD88 is associated withall TLRs, except TLR3, and phosphorylates IRAK, IRF5, IRF7, MAPK andNF-κB, which enhance the expression of type I IFN, inflammatory cytokineand IFN-inducible genes (Kawai and Akira, Semin. Immunol. 19:24-32(2007)). Unlike other TLRs, endosomal TLRs, TLR3, 7, 8 and 9, allrecognize microbial or host nucleic acids, as PAMPs or DAMPs,respectively. The redundancy and interconnectedness of the TLR signalingpathway suggests that it will be important to inhibit the activity ofmultiple TLRs simultaneously to effectively control inflammatory andautoimmune responses and to enhance the clinical efficacy of TLRantagonists as therapeutic agents.

It was discovered recently that certain cationic polymers are able tocounteract the activity of a variety of oligonucleotide-based drugs(e.g., aptamers), irrespective of their nucleotide sequences (Oney etal, Control of Aptamer Activity by Universal Antidotes: An Approach toSafer Therapeutics, Nature Medicine (in press)). Moreover, immunestimulatory siRNA, a TLR7 agonist, condensed with a cyclodextrin-basedpolymer has been shown not to activate TLR7 (Hu-Lieskovan et al, CancerRes. 65:8984-8992 (2005)). The present invention results, at least inpart, from studies designed to determine whether agents that bind DNAsand RNAs in a sequence-independent manner (e.g., nucleic acid-bindingcationic polymers) can neutralize endosomal TLR ligands and therebyinhibit activation of the corresponding TLRs.

SUMMARY OF THE INVENTION

The present invention relates generally to PRRs, including TLRs (e.g.,endosomal TLRs). More specifically, the invention relates to a method ofinhibiting nucleic acid-induced activation of, for example, endosomalTLRs using an agent that binds to the nucleic acid (“nucleic acidbinding agent”), preferably, in a manner that is independent of thenucleotide sequence, the chemistry (e.g., DNA or RNA, with or withoutbase or sugar modifications) and/or the structure (e.g., double-strandedor single-stranded, complexed or uncomplexed with, for example protein)of the nucleic acid responsible for inducing TLR activation. Theinvention further relates to methods of controlling inflammatory and/orautoimmune responses resulting from nucleic acid-induced receptor (e.g.endosomal TLR) activation using such a nucleic acid binding agent. Theinvention further relates to methods of identifying nucleic acid bindingagents suitable for use in such methods.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Cationic polymers inhibit nucleic acid inducedactivation of TLR3 and TLR9. (FIG. 1A) The murine macrophage cell line,Raw264.7 was co-incubated in a 24-well microplate with a TLR9 agonist(CpG) (2 μM), a TLR3 agonist (poly I:C) (10 μg/ml) or a TLR4 agonist(LPS) (100 ng/ml) along with the cationic polymers, CDP, HDMBr, PAMAM,poly L-lysine or protamine (20 μg/ml) or PBS. Unmethylated GpC ODNs wereused as a negative control for CpG. After 18-hours of incubation,culture supernatants were collected and analyzed for cytokines by ELISA.(FIG. 1B) The treated cells were tested for their expression of theco-stimulatory molecule CD86 using FACS. The light blue line representsPBS-treated cells. Green and red lines represent GpC- and CpG-treatedcells, respectively. Data represents three individual experiments. Errorbar is S.D.; n=3. *P<0.005 (both TNFα and IL-6; CpG or poly I:C+Cationicpolymers vs CpG or Poly I:C alone); ‡ P=0.0169 and 0.0395 (TNFα andIL-6, respectively; poly I:C+CDP vs poly I:C alone);

P=0.0256 and 0.0281 (TNFα and IL-6, respectively; poly I:C+protamine vspoly I:C alone).

FIGS. 2A and 2B. Timing of cationic polymer mediated inhibition of TLRactivation. (FIG. 2A) Cells were incubated with CpG (2 μM) in a 24-wellmicroplate. CDP (20 μg/ml) was added at 0, ½, 1, 2, 4, 8 or 12 hoursfollowing the addition of CpG. At 24 hours after CpG treatment culturesupernatants were collected and analyzed for TNFα and IL-6 production.(FIG. 2B) Cells were pre-incubated for 1 or 2 hours with CDP or PBS,washed three times with complete medium and then incubated in culturemedia supplemented with CpG. Simultaneous treatment of cells with CpGand CDP was used as a control. At 5 hours after CpG treatment the amountof TNFα in the culture supernatants were measured by ELISA. Error bar isS.D.; n=3. *P<0.0001 (both TNFα and IL-6; CpG+CDP vs CpG alone); ‡P=0.0230 and <0.0001 (TNFα and IL-6, respectively; CpG+CDP vs CpGalone);

P=0.0257 and 0.0003 (TNFα and IL-6, respectively; CpG+CDP vs CpG alone).

FIGS. 3A and 3B. Dose-dependent inhibition of cationic molecules on TLR3and TLR9 activation. 1×10⁶ Raw264.7 cells were cultured for 18 hourswith either CpG (1 μM) (FIG. 3A) or poly I:C (10 μg/ml) (FIG. 3B) in thepresence or absence of CDP (□), HDMBr (

) or PAMAM (▪) at the indicated concentration. Amounts of TNFα and IL-6in the culture supernatant were measured by ELISA. Error bar is S.D.;n=3. NT: not tested.

FIGS. 4A-4C. TLR3- or TLR9-mediated acute liver inflammation can bealleviated by nucleic acid-binding polymers. (FIG. 4A) Mice (5-10mice/group) were i.p. injected with D-GalN (20 mg) alone, CpG (51 μg)alone, D-GalN+GpC (51 μg) or D-GalN+CpG (51 μg). After 5-10 minutes, PBS(100 μl), CDP (200 μg; blue diamond), HDMBr (200 or 400 μg; redtriangle) or PAMAM (200 or 400 μg; green rectangle) was administeredi.p. into mice challenged with D-GalN+CpG. Mice were monitored daily forsurvival. (FIG. 4B) Mixture of Poly I:C (200 μg) and D-GalN (20 mg) inPBS (100 μl) was injected i.p. into mouse (5 mice/group). Subsequently,PBS (100 μl; black circle), CDP (400 or 800 μg; blue diamond), HDMBr(200 or 400 μg; red triangle) or PAMAM (200 or 400 μg; green rectangle)was injected i.p. There is 5-10 minutes interval between injections.(FIG. 4C) Mice were injected with PBS, CpG+D-GalN or CpG+D-GalN+CDP.Sixteen hours following injection, liver specimens were collected forhistological studies (hematoxylin and eosin staining). A representativeof three individual results. Magnification ×20.

FIGS. 5A and 5B. Stoichiometry of TLR inhibition of CDP. Raw264.7 cellswere cultured for 18 hours with either CpG (1 μM, 2 μM, 4 μM, 8 μM)(FIG. 5A) or poly I:C (10 μg/ml or 25 μg/ml) (FIG. 5B). TLR ligands weresimultaneously supplemented with CDP at various concentration (0, 4, 8,12, 16, 20, 24, 36, 48 μg/ml for CpG; 0, 10, 20, 30, 40, 80, 160 μg/mlfor poly I:C). Amount of TNFα was measured by ELISA. % inhibition wascalculated by ([CpG or poly I:C]−[CpG or poly I:C+CDP])/[CpG or polyI:C]×100.

FIG. 6. Cellular toxicity of cationic molecules. 1×10⁶ Raw264.7 cellswere cultured for 24 hours with CDP (black), HDMBr (red), PAMAM (blue),PPA-DPA (green), protamine (gray) or poly L-lysine (purple) at variousconcentration (10, 20, 40, 80, 160, 280, 400 and 600 μg/ml). Viabilityof cells was analyzed using hematocytometer after staining with trypanblue (Sigma, St. Louis, Mo.).

FIGS. 7A-7D. CDP enhanced CpG uptake of cells. Raw264.7 cells (1×10⁵cells/well) were cultured overnight in 8-well chamber slide (Nalge NuncInternational Corp, Naperville, Ill.). After thrice washing with coldcomplete media, cells were replenished with fresh complete mediaincluding 1 μM of CpG conjugated with 6-FAM at 5′ end with or without 10μg/ml of CDP. Cells were incubated for 1 (FIGS. 7A and 7C) or 2 hours(FIGS. 7B and 7D) at either 4° C. or 37° C. Fluoresce signals wereobserved with the Olympus IX71 Inverted Microscope (Olympus, CenterValley, Pa.). The images were analyzed using the Olympus DP ControllerVer.1.2.1.108. Data represents two individual experiments. Magnificationis 40×.

DETAILED DESCRIPTION OF THE INVENTION

PRRs are a pivotal component of host immune cells to protect tissuesfrom various harmful stimuli, such as pathogens and damaged cells. Avariety of PRRs, including RIG-I-like receptors (RLRs), dsRNA-dependentprotein kinase R (PKR), DNA-dependent activator of IRFs (DAI) and TLRscan recognize diverse products of pathogens and damaged cells that arereferred to PAMPs and DAMPs (Lotze et al, Immunol. Reviews 220:60-81(2007)).

TLRs play a central role in host innate and acquired immunity, as wellas in the pathogenesis of various diseases, including infectiousdiseases, inflammatory diseases and autoimmune diseases. TLRs 3, 7, 8and 9 are localized in endosomes can be activated by microbial and hostnucleic acids.

The present invention relates, in one embodiment, to a method ofinhibiting nucleic acid-induced activation of endosomal TLRs. The methodcomprises administering to a patient in need thereof an agent that bindsnucleic acids responsible for induction of TLR activation in an amountand under conditions such that inhibition of that activation iseffected. Advantageously, the agent binds the nucleic acids in a mannerthat is independent of the nucleotide sequence, the chemistry (e.g., DNAor RNA, with or without base or sugar modifications) and/or thestructure (e.g., double-stranded or single-stranded, complexed oruncomplexed with, for example, a protein) of the nucleic acidsresponsible for inducing TLR activation. The present method can be usedto treat inflammatory and/or autoimmune responses resulting fromendosomal activation.

Nucleic acid binding (scavenging) agents of the invention includepharmaceutically acceptable member(s) of a group of positively chargedcompounds, including proteins, lipids, and natural and syntheticpolymers, that can bind nucleic acids in, for example, biologicallyfluids.

Proteinaceous nucleic acid binding agents of the invention includeprotamines, a group of proteins that yield basic amino acids onhydrolysis and that occur combined with nucleic acid in the sperm offish, such as salmon. Protamines are soluble in water, are notcoagulated by heat, and comprise arginine, alanine and serine (most alsocontain proline and valine and many contain glycine and isoleucine). Inpurified form, protamine has been used for decades to neutralize theanticoagulant effects of heparin. Nucleic acid binding agents of theinvention also include protamine variants (e.g., the +18RGD vàriant(Wakefield et al, J. Surg. Res. 63:280 (1996)) and modified forms ofprotamine, including those described in Published U.S. Application No.20040121443. Other nucleic acid binding agents of the invention includeprotamine fragments, such as those described in U.S. Pat. No. 6,624,141and U.S. Published Application No. 20050101532. Nucleic acid bindingagents of the invention also include, generally, peptides that modulatethe activity of heparin, other glycosaminoglycans or proteoglycans (see,for example, U.S. Pat. No. 5,919,761). The invention further includespharmaceutically acceptable salts of the above-described nucleic acidbinding agents, as appropriate, including sulfate salts.

Proteinaceous nucleic acid binding agents of the invention also includeDNA and/or RNA reactive antibodies. For example, anti-nuclearantibodies, such as those indicative of lupus erythematosis, Sjögren'ssyndrome, rheumatoid arthritis, autoimmune hepatitis, scleroderma,polymyositis and dermatomyositis, can be used. Specific examples ofantibodies that recognize RNA/DNA include those described by Kitagawa etal (Mol. Immunol. 19(3):413-20 (1982)), Boguslawski et al (J. Immunol.Methods 89(1):123-30 (1986)), Williamson et al (Proc. Natl. Acad. Sci.98(4):1793-98 (2001)), and Blanco et al (Clin. Exp. Immunol. 86(1):66-70(1991)).

In addition, heterogeneous nuclear ribonucleoproteins (HNRPs) can alsobe used in accordance with the invention. Cationic, peptides that bindnucleic acids (e.g., in a sequence-independent manner) are suitable foruse. For example, a chimeric peptide synthesized by adding nonamerarginine residues at the carboxy terminus of RVG (to yield RVG-9R) hasbeen described by Kumar et al (Nature 448:39-43 (2007)). Viral proteinsthat package (e.g., coat) DNA or RNA (e.g., HIV gag protein), andpeptides derived therefrom, can also be used in the present methods.

Cationic lipids can also be used as nucleic acid binding agents inaccordance with the invention. Suitable cationic lipids include thosedescribed by Morille et al (Biomaterials 29:3477 (2008)) (e.g., linearpoly(ethyleneimine) (PEI), poly(L-lysine) (PLL), poly(amidoamine)(PAMAM) dendrimer generation 4, chitosan, DOTMA, DOTAP, DMRIE, DOTIM,DOGS, DC-Choi, BGTC and DOPE).

Nucleic acid binding agents of the invention also include intercalatingagents. Examples include ethidium bromide, proflavine, daunomycin,doxorubicin and thalidomide. Nucleic acid binding porphyrins can also beused in accordance with the invention (see Table 1).

Preferred nucleic acid binding agents of the invention includepolycationic polymers. Preferred polycationic polymers includebiocompatible polymers (that is, polymers that do not cause significantundesired physiological reactions) that can be either biodegradable ornon-biodegradable polymers or blends or copolymers thereof. Examples ofsuch polymers include, but are not limited to, polycationicbiodegradable polyphosphoramidates, polyamines having amine groups oneither the polymer backbone or the polymer side chains, nonpeptidepolyamines such as poly(aminostyrene), poly(aminoacrylate),poly(N-methyl aminoacrylate), poly(N-ethylaminoacrylate),poly(N,N-dimethyl aminoacrylate), poly(N,N-diethylaminoacrylate),poly(aminomethacrylate), poly(N-methyl amino-methacrylate), poly(N-ethylaminomethacrylate), poly(N,N-dimethyl aminomethacrylate),poly(N,N-diethyl aminomethacrylate), poly(ethyleneimine), polymers ofquaternary amines, such as poly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride); natural orsynthetic polysaccharides such as chitosan, cyclodextrin-containingpolymers, degradable polycations such aspoly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA); polycationicpolyurethanes, polyethers, polyesters, polyamides, polybrene, etc.Particularly preferred cationic polymers include CDP, CDP-im, PPA-DPA,PAMAM and HDMBr.

Nucleic acid binding agents of the invention can include compounds oftypes described in Table 1, or derivatives thereof. Several of thecompounds described in Table 1 contain cationic-NH groups permittingstabilizing charge-charge interactions with a phosphodiester backbone.Nucleic acid binding agents of the invention containing secondary aminescan include, for example, 5-350 such groups (e.g., 5-300, 5-250, 5-200,5-100, 5-50, 50-100, 50-200, 50-300, 50-350, 100-200, 100-300, 100-350,200-350, 200-300, or 250-350), and can have a molecular weight in therange of, for example, 2,000 to 50,000 (e.g., 10,000 to 50,000 or 20,000to 40,000).

TABLE 1 Compound Abbreviation Molecular structure Remark Poly-L-lysinePLL

1. Commercially available. 2. Carbonyl moiety (—C═O) which could permitadditional stabilization to the complex through hydrogen bonds with DNA.Poly-L-ornithine PLO

1. Commercially available. 2. Carbonyl moiety (—C═O) which could permitadditional stabilization to the complex through hydrogen bonds with DNA.Polyphosphoramidate polymer series PPA-SP PPA-BA PPA-EA PPA-MEA PPA-DMA

PPA 1. Polymers with an identical backbone but different side chainsranging from primary to PPA-DEA PPA-TMA PPA-DPA

PPA-SP quaternary amines. Provide a platform for a systematic study2.Lower cytotoxicity —NH—(CH₂)₄—NH₂ PPA-BA compared with —NH—(CH₂)₂—NH₂PPA-EA polyethylenimine —NH—(CH₂)₂—NH—CH₃ PPA-MEA (PEI) and poly-L-

PPA-DMA lysine (PLL).

PPA-DEA

PPA-TMA

PPA-DPA Polyphosphoramidate diprophylamine- poly ethylene glycolcopolymer PPA-DPA-b- PEG₂₀₀₀

1. a copolymer of PPA-DPA and PEG. Polyethyleneimine PEI

1. Commercially available. 2. PEI with branched structure condenses DNAto a greater extent than linear ones. 3. high cytotoxicity. Ionene e.g.polybrene

1. Commercially available. 2. Have high charge density. Naturalpolyamine H₂N—(CH₂)₄—NH₂ 1. Commercially e.g.H₂N—(CH₂)₃—NH—(CH₂)₄—NH—(CH₂)₃—NH₂ available. PutrescineH₂N—(CH₂)₄—NH—(CH₂)₃—NH₂ 2. The most extensive Spermine work Spermidineon their binding with DNA has been carried out and have remarkableeffects on the DNA condensation. Poly(allylamine) PAL

1. Commercially available. 2. Highly positive charged 3. Low toxicity.Peptide nucleic acid PNA

1. Commercially available. 2. Binding through Watson-crick base pairing,thus binding is typically stronger and more rapid. Water solubleporphyrin e.g. poly tetra(p- aminophenyl) porphyrin poly tetra(methylpyridine) porphyrin H₂TAPP H₂TMPyP₄

1. Commerically available. 2. One or two —N⁺(CH₃)₃ groups of one TAPPmolecule bind with the phosphate groups. 3. The stacking of TAPP alongthe surface of DNA leads to a favorite binding. 4. Especially goodbinding with G- quadruplex through pi- pi interaction.

Poly(porphyrin) or Porphyrin ladder e.g. poly (H₂ (p-TAPP) poly(por)A-AN))

Poly (N,N- dimethylacrylamide) PDMA

Poly (2- Methacryloyloxyethyl phosphorylcholine) PMPC

Dendrimers e.g. polyamidoamine dendrimer PAMAM Dendrimer G2

1. Commercially available. 2. Branched spherical shape and a highdensity surface charge. 3. Low cytotoxicity. e.g. polypropyl- eneiminedendrimer PPI dendrimer

1. A class of amine- terminated polymers, demonstrated to be efficientgene delivery vectors. 2. Low cytotoxicity in a wide range of mammaliancell lines. 3. Unique molecular structures, with defined molecularweight, surface charge and surface functionality. These properties ofdendrimers provide a platform for a systematic study. Partiallydeacetylated Chitin

1. Commercially available. Cyclodextrin grafted branched PEI or linearPEI CD-bPEI CD-lPEI

1. Their IC₅₀'s are 2-3 orders of magnitude (α-CD: six sugar ring β-CD:seven sugar ring γ-CD: eight sugar ring)

higher than the corresponding non- cyclodextrin-based polymer.

Cyclodextrin Containing Polymers CDP

CDP-Im

Advantageously, the binding affinity of a nucleic acid binding agent ofthe invention for a nucleic acid, expressed in terms of Kd, is in the pMto μM range, preferably, less than or equal to 50 nM; expressed in termsof binding constant (K), the binding affinity is advantageously equal toor greater than 10⁵M⁻¹, preferably, 10⁵M⁻¹ to 10⁸M⁻¹, more preferably,equal to or greater than 10⁶M⁻¹. Thus, the binding affinity of thesequence-independent nucleic acid binding agents can be, for example,about 1×10⁵M⁻¹, 5×10⁵ M⁻¹, 1×10⁶M⁻¹, 5×10⁶M⁻¹, 1×10⁷ M⁻¹, 5×10⁷ M⁻¹; orabout 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM. “K” and“Kd” can be determined by methods known in the art, including surfaceplasmon resonance or a real time binding assay such as Biacore.

Preferred nucleic acid binding agents of the invention simultaneouslylimit the activation of multiple endosomal TLRs (e.g., TLR3 and TLR9).Particularly preferred are CDP or CDP-im, HDMBr and PAMAM (see U.S. Pat.Nos. 7,270,808, 7,166,302, 7,091,192, 7,018,609, 6,884,789, 6,509,323,5,608,015, 5,276,088, 5,855,900, U.S. Published Appln. Nos. 20060263435,20050256071, 200550136430, 20040109888, 20040063654, 20030157030, Daviset al, Current Med. Chem. 11(2) 179-197 (2004), and ComprehensiveSupramolecular Chemistry vol. 3, J. L. Atwood et al, eds, Pergamon Press(1996)).

As indicated above, the present invention provides a method ofcontrolling (inhibiting or preventing) autoimmune and/or inflammatoryresponses associated with activation of TLRs (e.g., endosomal TLRs suchas TLR3 and TLR9). Such responses play a role in the pathogensis ofdiseases/disorders that are associated with presence in the circulationof the patient of free nucleic acids, either pathogen-derived (e.g.,viral- or bacterial-derived) nucleic acids or nucleic acids releasedfrom dead or damaged host cells. Specific diseases/disorders that can betreated using nucleic acid binding agents of the invention includeinfectious diseases, cardiovascular disease, cancer, bacterial sepsis,multiple sclerosis, systemic lupus erythematosis, rheumatoid arthritis,COPD, obesity and psoriasis.

RLRs are a family of cytoplasmic RNA helicases includingretinoic-acid-inducible protein I (RIG-I) andmelanoma-differentiation-associated gene 5 (MDA5). RIG-I recognizeuncapped 5′-triphosphate ssRNA and short dsRNA while MDA5 recognize longdsRNA (Pichlmair et al, Science 314:997-1001 (2006), Hornung et al,Science 314:994-997 (2006), Kato et al, J. Exp. Med. 205:1601-1610(2008)). Signaling of RLRs is initiated by interaction of caspaserecruitment domain (CARD)-containing adapter molecule, IFNβ promoterstimulator-1 (IPS-1), and induce production of type I IFN andinflammatory cytokines (Kawai et al, Ann. N.Y. Acad. Sc. 1143:1-20(2008)). PKR is an IFN-inducible cytosolic enzyme and recognizes viraldsRNAs while DAI recognizes cytoplasmic dsDNA (Langland et al, Virus Res119:100-110 (2006), Takaoka et al, Nature 448:501-505 (2007)). Thesecytoplasmic PRRs, including RIG-I, MDA5 and PKR, are able to recognizeRNAs or DNAs and activation of these PRRs is associated with type I IFNproduction. Although their involvement in the pathogenesis ofinflammatory and autoimmune diseases has not been fully elucidated, thecytoplasmic nucleic acid-sensing PRRs may also contribute to thepathogenesis of such diseases because signaling from these receptorsrobustly elicits production of IFNα, one of the major pathogenic factorsin a variety of inflammatory diseases (J. Banchereau et al, Immunity20:539-550 (2004)). Therefore, the present invention also relates amethod of inhibiting nucleic-acid induced activation of these members ofthe PRR family using the approaches and agents described above.

Another application of nucleic acid-binding agents (scavengers)described herein is to counteract the effects of DNA and RNA moleculesthat are released from cells and subsequently induce thrombosis(Kannemeier et al, Proc. Natl. Acad. Sci. 104:6388-6393 (2007); Fuchs etal, Proc. Natl. Aad. Sci. Published Online before Print Aug. 23, 2010).Recently it has been observed that RNA and DNA molecules can activatethe coagulation pathway as well as platelets and thereby engender bloodclotting (Kannemeier et al, Proc. Natl. Acad. Sci. 104:6388-6393 (2007);Fuchs et al, Proc. Natl. Acad. Sci. Published Online before Print Aug.23, 2010). Since nucleic acid binding agents (scavengers) describedherein can bind RNA and DNA molecules and shield them from otherpotential binding partners, such agents can be employed to inhibit theability of DNA and RNA molecules to bind and activate coagulationfactors and platelets. In so doing, these RNA/DNA scavengers can beutilized to limit nucleic acid-induced pathological blood coagulation.Thus nucleic acid binding agents (scavengers) described herein representnovel entities for preventing the induction and progression of a varietyof thrombotic disorders including myocardial infarction, stroke and deepvein thrombosis.

The nucleic acid binding agents of the invention, or pharmaceuticallyacceptable salts thereof, can be administered to the patient via anyroute such that effective levels are achieved in, for example, thebloodstream. The optimum dosing regimen will depend, for example, on thenucleic acid binding agent, the patient and the effect sought.Typically, the nucleic acid binding agent will be administered orally,transdermally, IV, IM, IP or SC. The nucleic acid binding agent can alsobe administered, for example, directly to a target site, for example,directly to a tumor (e.g., a brain tumor) when cancer is the disease tobe treated. Advantageously, the nucleic acid binding agent isadministered as soon as clinical symptoms appear and administration isrepeated as needed.

The nucleic acid binding agents (including nucleic acid binding polymersincorporated into microparticles or nanoparticles or beads), orpharmaceutically acceptable salts thereof, can be formulated with acarrier, diluent or excipient to yield a pharmaceutical composition. Theprecise nature of the compositions of the invention will depend, atleast in part, on the nature of the nucleic acid binding agent and theroute of administration. Optimum dosing regimens can be readilyestablished by one skilled in the art and can vary with the nucleic acidbinding agent, the patient and the effect sought.

Proteinaceous nucleic acid binding agents of the invention can also beproduced in vivo following administration of a construct comprising asequence encoding the proteinaceous nucleic acid binding agent(Harrison, Blood Rev. 19(2):111-23 (2005)).

It will be appreciated that the treatment methods of the presentinvention are useful in the fields of both human medicine and veterinarymedicine. Thus, the patient (subject) to be treated can be a mammalpreferably a human. For veterinary purposes the subject can be, forexample, a farm animal such as a cow, pig, horse, goat or sheep, or acompanion animal such as a dog or a cat.

The invention also relates to methods of identifying nucleic acidbinding agents suitable for use in the above-described methods. In oneembodiment, endosomal TLR-containing cells, preferably, mammalian cells(e.g., mammalian macrophage cells in culture), are incubated with afirst endosomal TLR agonist (e.g., CpG DNA or single or double strandedRNA or nucleic acid-containing particles) in the presence and absence ofa test agent. Following incubation, a culture supernatant sample can betaken and analyzed for the presence of a product of an intracellularsignaling event initiated by TLR activation, for example, one or morecytokines (e.g., TNFα and/or IL-6). These steps can be repeated with anendosomal TLR agonist having a sequence, chemistry and/or structuredifferent from that of the first agonist. A test agent that inhibitsendosomal TLR agonist activation, preferably, in a manner independent ofthe sequence, chemistry and/or structure of the endosomal TLR agonistused, (that inhibition of activation being evidenced by inhibition ofproduction of a product of an intracellular signaling event initiated byTLR activation (e.g., cytokine production) (e.g., in a dose dependentmanner)) can then be tested in vivo, for example, in mice, to furtherassess its suitability for use in the methods described herein.

Certain aspects of the invention can be described in greater detail inthe non-limiting Example that follows.

EXAMPLE

Experimental Details

Animal and Cell Line Studies.

8-9 weeks old C57BL/6 mice were purchased from the Jackson Laboratory(Bar Harbor, Me.). The murine macrophage cell line, Raw264.7 wasobtained from ATCC (Manassas, Va.).

Cytokine Production of Murine Macrophage.

1×10⁶ Raw264.7 cells were cultured in complete medium including DMEMwith 10% heat-inactivated. FBS, penicillin, streptomycin and L-glutamine(2 mM) (all from Invitrogen, Carlsbad, Calif.) in a 24-well cultureplate at 37° C. in a humidified atmosphere with 5% CO₂. To study TLRactivation, the complete medium was supplemented with phosphorothioateB-type CpG DNA 1668 (5′-TCCATGACGTTCCTGATGCT-3′), a phosphorothioate GpCDNA 1720 (5′-TCCATGAGCTTCCTGATGCT-3′) as a control CpG DNA (both fromIDT, Coralville, Iowa) or a mimetic of viral dsRNA, poly I:C(Amersham/GE Healthcare, Piscataway, N.J.) at various concentrations.Bacterial LPS serotype 026:B6 (100 ng/ml) (Sigma-Aldrich, Saint Louis,Mo.) activating TLR4 were used as a non-nucleotide-based TLR ligand. Toblock TLR activation CDP (Calando Pharmaceuticals, Pasadena, Calif.),protamine (APP, Schaumburg, Ill.), PPA-DPA, PAMAM, poly-L-lysine orHDMBr (polybrene) (kindly provided by Dr. Kam W. Leong, Duke University,Durham, N.C.) at various concentrations were simultaneously treated witheither CpG DNA or poly IC. After 18 hours of incubation, culturesupernatant were collected and stored at −80° C. for later analyses. Theproduction of TNFα and IL-6 were analyzed with BD OptEIA™ ELISA sets (BDBiosciences, Franklin Lakes, N.J.) by following the manufacturer'sinstructions.

Co-Stimulatory Molecule Expression on Macrophage.

1×10⁶ Raw264.7 cells were cultured with phosphate buffer saline (PBS),GpC DNA (2 μM) or CpG DNA (2 μM). To block binding of CpG DNA and TLR9CDP, HDMBr, PAMAM, PPA-DPA, poly-L-lysine or protamine (20 μg/ml each)were co-treated with CpG DNA. After 18 hours, cells were detached fromplates by treatment of 0.05% Trypsin-EDTA (Invitrogen), washed twicewith PBS and stained with either phycoerythrin (PE)-anti-mouse CD86(GL1) or PE-rat IgG2a, κ as an isotype control (all from eBioscience,San Diego, Calif.). Cells were washed with PBS, fixed with 4%formaldehyde, and analyzed on a FACS Caliber (BD Biosciences).

Mouse TLR-Mediated Acute Liver Injury.

TLR3 or TLR9-mediated acute liver injury in a D-galactosamine-sensitizedmice was performed as previously described (Alexopoulou et al Nature413:732-738 (2001), Duramad et al, J. Immunol. 174:5193-5200 (2005)).Briefly, C57BL/6 mice were injected intraperitoneally (i.p.) with PBS(100 μl), CpG DNA (25 to 51 μg), GpC DNA (50 μg) or poly I:C (50 to 200μg) with or without D(+)Galactosamine, Hydrochloride (D-GalN) (EMDBiosciences, La Jolla, Calif.) (20 mg). Five to ten minutes after toxinchallenge, cationic molecules (200 to 800 μg) were injected i.p.Viability of mice was monitored for a week.

Histopatholoy.

Liver lobes were excised from mice 24 hours after injection ofCpG+D-GalN with or without cationic molecules. The liver specimens werefixed with 4% formaldehyde, embedded in OCT and sectioned at a thicknessof 20 μm before staining with hematoxylin and eosin for lightmicroscopic examination.

Statistical Analysis.

The difference of cytokine production among experimental groups wascompared by the paired two-tailed Student's t test analyzed withMicrosoft Office Excel 2003. Significance of survival was determined bythe log-rank test analyzed with GraphPad Prism® Version 4.0b. Aprobability of less than 0.05 (P<0.05) was used for statisticalsignificance.

Results

Six agents known to bind nucleic acids were evaluated for their abilityto attenuate endosomal TLR responses: β-cyclodextrin-containingpolycation (CDP), polyphosphoramidate polymer (PPA-DPA), polyamidoaminedendrimer, 1,4-diaminobutane core, G3 (PAMAM), poly-L-lysine,hexadimethrine bromide (HDMBr; also known as polybrene) and protamine.Five of the compounds inhibited polyinosinic-polycytidylic acids (polyI:Cs), a dsRNA activator of TLR3, stimulation of macrophages as measuredby TNFα and IL-6 production and three prevented inflammatory cytokineproduction from the cells stimulated with unmethylated CpG DNA, a TLR9agonist (FIG. 1A). The CpG DNA-inhibitory cationic polymers also impededthe up-regulation of co-stimulatory molecules expressed on macrophages(FIG. 1B). Interestingly, the inhibitors could be administered up to 4hours after the CpG DNA and still significantly reduce TNFα and IL-6production from macrophages (FIG. 2). Pre-treatment of macrophages withCDP, however, did not alter the ability of the cells to produceinflammatory cytokines (FIG. 2). By contrast, the nucleic acid-bindingcationic polymers did not inhibit LPS-mediated activation ofmacrophages, which indicates that they specifically interfere withrecognition of nucleic acids by TLRs.

The nucleic acid-binding polymers inhibit TLR3 and TLR9 activation in adose-dependent manner. A dose-escalation study demonstrated that 8 to 12μg/ml of the polymers, CDP, HDMBr and PAMAM, which inhibited theactivation of both TLR3 and TLR9, can inhibit inflammatory cytokineproduction by greater than 95% from macrophages treated with CpG DNAs (1μM) and 5 to 40 μg/ml of these same polymers can reduce cytokineproduction by greater than 95% from cells treated with poly I:C (10μg/ml) (FIGS. 3A and 5).

One concern about using cationic polymers as therapeutic agents is theirpotential toxicity since certain cationic carriers are know to have highcytotoxicity (Hunter, Adv. Drug Deliv. Rev. 58:1523-1531 (2006)). PolyL-lysine (10-40 μg/ml) has been shown to induce significant apoptosis ofmammalian cells (Symonds et al, FEBS Lett. 579:6191-6198 (2005)). Bycontrast, the LD₅₀ of CDP is 200 mg/kg in mice (Hwang et al, Bioconjug.Chem. 12:280-290 (2001)). Therefore, the cytotoxicity of the cationicpolymers used in the current study was evaluated on macrophages (FIG.6). Poly L-lysine and PPA-DPA induced over 50% cell death atapproximately 20 and 40 μg/ml, respectively, while PAMAM, protamine andHDMBr induced over 50% cell death at about 160, 280 and 600 μg/ml,respectively. The CDP polymer was well tolerated on macrophages. In miceinjected with the CDP, HDMBr and PAMAM at 40 mg/kg, no adverse effectson viability were observed (data not shown). In summary, poly L-lysineand PPA-DPA have a relatively high cytotoxicity while PAMAM, HDMBr andCDP have much less toxicity in vitro and in vivo.

Finally, the ability of the nucleic acid-binding polymers to limitendosomal TLR activation in vivo was evaluated. It has been shown thatinjection of CpG DNA or poly I:C into mice sensitized withD-galactosamine (D-GalN) induces a TLR-mediated acute inflammatoryresponse which can result in liver damage and death (Alexopoulou et al,Nature 413:732-738 (2001), Duramad et al, J. Immunol. 174:5193-5200(2005)). Consistent with previous reports, greater than 90% of the micedied by 48 hours following administration of D-GalN and CpG DNA or polyI:C while none of the mice injected with D-GalN alone, CpG DNA alone orD-GalN and control GpC DNA died. Strikingly, administration of one ofthree different nucleic acid-binding polymers, CDP, HDMBr or PAMAM,immediately following D-GalN and CpG DNA or poly I:C resulted insignificant protection of the animals in a dose-dependent manner andreduced mortality by almost 100% in several cases (FIGS. 4A and 4B).Histological examination of livers from treated mice also demonstratedthat inflammation and associated hemorrhage were greatly reduced in thepolymer treated animals (FIG. 4C).

Cationic polymers are commonly used for gene or siRNA delivery and aredesigned to facilitate cellular internalization and endosomal escape(Morille et al, Biomaterials. 29:3477-3496 (2008)). Because they trafficthrough the endosomal compartment, cationic lipids have been used todeliver siRNAs and immune stimulatory ssRNAs to activate endosomal TLR7or TLR8 (Judge et al, Nat. Biotechnol. 23:457-462 (2005), Sioud, J. Mol.Biol. 348:1079-1090 (2005)). Moreover, synthetic ssRNAs or mRNAspre-condensed with protamine induced inflammatory cytokine production inhuman PBMCs via activation of TLR7 or TLR8 (Scheel et al, Eur. J.Inununol. 35:1557-1566 (2005)). Similarly, it was observed thattreatment with protamine did not block but significantly enhancedinflammatory cytokine production from cells stimulated with poly I:C(FIG. 1A). In striking contrast, it was observed in the above-describedstudies that the cationic polymers, CDP, HDMBr and PAMAM, neutralize theability of nucleic acid-based TLR3 and TLR9 ligands to activate theircognate TLRs and induce inflammatory responses. Several potentialexplanations exist for these observed differences. In the presentstudies, cells were treated with endosomal TLR ligands and cationicpolymers separately while in the previous studies immune stimulatoryRNAs were pre-condensed with cationic molecules before exposure tocells. Thus, the pre-condensation of RNA and cationic molecules couldgenerate a particle that might be efficiently endocytosed. By contrast,nucleic acids, that are not assembled into particles, may be only poorlytaken up by cells and addition of the polymers would form smallcomplexes not recognized by the cell. To test this possibility, thecellular uptake of CpG DNAs was evaluated. Unexpectedly, treatment withCDP enhanced cellular uptake of CpG DNAs, even though this did not leadto endosomal TLR9 activation (FIG. 7). The reason why CpG delivered intocells in this manner does not elicit a TLR response is unclear. Thepolymer may alter endosomal maturation and thus TLR signaling or thepolymer may directs the CpG into a distinct intracellular traffickingpathway (Morille et al, biomaterials 29:3477-3496 (2008), Krieg, Annu.Rev. Immunol. 20:709-760 (2002), Jozefowski et al, J. Leukoc. Biol.80:870-879 (2006)). Further investigation will be required to understandhow cationic polymers neutralize nucleic acid activation of endosomalTLRs and why some cationic polymers are more effective than others atimpeding such responses.

In summary, nucleic acid-binding polymers can simultaneously limit theactivation of multiple endosomal TLRs. As such, these polymers representpromising therapeutic agents for treating patients with inflammatorydiseases and autoimmune diseases. Additional preclinical and clinicalstudies will evaluate this possibility.

All documents and other information sources cited above are herebyincorporated in their entirety by reference.

What is claimed is:
 1. A method of inhibiting nucleic acid-inducedactivation of toll-like receptor 3 (TLR3) or toll-like receptor 9 (TLR9)to treat a TLR3 or TLR9 induced inflammatory or immune responsecomprising administering to a patient in need of said inhibition ofnucleic acid-induced activation of TLR3 or TLR9 an agent that binds anucleic acid responsible for said induction of activation in an amountand under conditions such that said inhibition of said activation iseffected, wherein the agent is poly(amidoamine) (PAMAM).
 2. The methodaccording to claim 1 wherein said agent binds said nucleic acid in amanner that is independent of nucleotide sequence, chemistry orstructure.
 3. The method of claim 1, further comprising exposing thepatient to a nucleic acid prior to administering the agent.
 4. Themethod of claim 1, wherein the patient was exposed to a nucleic acidprior to administration of the agent.
 5. The method of claim 4, whereinthe nucleic acid is pathogen-derived or is released from dead or damagedcells of the patient.
 6. The method of claim 1, further comprisingdetecting the inhibition of activation of TLR3 or TLR9 by measuringTNF-α or IL-6 production in the patient.
 7. The method of claim 1,further comprising detecting the inhibition of activation of TLR3 orTLR9 by measuring CD86 expression.
 8. The method of claim 1, whereinadministration of the agent results in a reduction in the acuteinflammatory response in the patient.
 9. The method of claim 1, whereinthe agent does not affect lipopolysaccharide-mediated inflammation. 10.The method of claim 1, wherein the patient suffers from a diseaseselected from the group consisting of an infectious disease, acardiovascular disease, cancer bacterial sepsis, multiple sclerosis,systemic lupus erythematosus, rheumatoid arthritis, chronic obstructivepulmonary disease, obesity and psoriasis.